N-terminal toxin signal peptides efficiently load therapeutics into a natural nano-injection system

Targeted delivery of therapeutics to specific cells is a major bottleneck towards personalized medicine. The extracellular injection system (eCIS) of Serratia entomophila, the antifeeding prophage (Afp), promises potential for drug delivery purposes. However, the precise mechanism of action, toxin location, and Afp loading remain unclear. Here, we reveal a minimal N-terminal signal peptide (NtSP) of the toxin Afp18, that plays a key role in toxin packing. By engineering fusion proteins, we demonstrate that Afp18's NtSP can shuttle effectors for Afp loading. We packed non-eCIS effectors, including CRISPR-Cas protein CasΦ-2 from Biggiephage, and a human antimicrobial peptide, LL37, into Afp. Additionally, NtSPs from eCIS effectors of other species facilitate loading of CasΦ-2 into Afp. We observed cargo being packed inside the Afp tail tube through cryo-EM single particle analysis. The presented results enhance our understanding of eCIS toxin packing and contribute to their development as targeted delivery systems. ### Competing Interest Statement Eva Maria Steiner-Rebrova and Nicholas M.I. Taylor filed a patent application related to this work (PCT/EP2023/068102). The other authors declare no competing interests.